It is well known that the determination of the presence of an antigen or antibody in body fluids has great significance in the diagnosis or remedy of diseases or abnormal or pathological states or conditions. Recently, certain antigen-antibody complexes or conjugates associated closely with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and certain glomerular nephritis have been found. Accordingly, it has currently become more significant to determine an antigen, antibody, or antigen-antibody complex or conjugate in body fluids to aid in the diagnosis or remedy of physiological or patholigical states or conditions in both human beings and animals.
It will be understood that the terms "anitgen or antibody" and "antigens or antibodies" when employed herein include an "antigen-antibody complex" and "antigen-antibody complexes" unless otherwise specifically noted to the contrary. It also will be understood that the terms "antigen-antibody complex" and "antigen-antibody complexes" when employed herein include an "antigen-antibody conjugate" and "antigen-antibody conjugates" unless otherwise stated.
One of the conventional methods for determining antigens, antibodies, or antigen-antibody complexes is the so-called slide-type method. This method consists of two procedures. The first procedure is to bind a substance that is specifically bindable to an antigen or antibody onto the surface of a carrier that is composed of fine-grained insoluble particles and suspend the bound substance in a suitable liquid. The second procedure is to mix the suspension, which is prepared in the first step, with a sample solution that contains the antigen or antibody to be determined. With this method, various kinds of antigens or antibodies can be determined semi-quantitatively by observing the agglutination of the suspension.
There are also methods known for quantitatively determining an antigen or antibody to be determined in a sample solution by spectrophotometrically or electrically measuring the results of the agglutination reaction, using the same materials as used in the slide-type method (Croatica Chemica Acta, 42, 457-466 (1970); European Journal of Biochemistry, 20, 553-560 (1971); Japanese Patent Application (Lay-Open) No. 24,015/1978; Japanese Patent Application (Lay-Open) No. 69,824/1978).
Of these quantitative measuring methods, the method for spectrophotometrically measuring the results of the agglutination reaction is outlined as follows: A substance specifically bindable to an antigen or antibody to be determined, such as, for example, the antigen or antibody corresponding to the antigen or antibody to be determined, is physically or chemically bound to finely divdided, insoluble carrier particles to provide sensitized carrier particles which are then suspended in an appropriate liquid and reacted with a sample solution containing the antigen or antibody to be determined under predetermined conditions, and the reaction mixture is measured with respect to its absorbancy or turbidity to quantitatively determine the antigen or antibody to be determined present in the sample solution. It will be understood that the terms "sensitized carrier particles" or "sensitized carrier particle" referred to herein mean the insoluble carrier particle sensitized by binding thereto such a substance as being specifically bindable to the antigen or antibody to be determined.
The afore-mentioned spectrophotometric method, however, encounters difficulties in providing an accurate quantitative determination of the antigen or antibody. In this method, the absorbancy or turbidity of a reaction mixture is measured while the reaction is still proceeding therein so that the absorbancy or turbidity thereof will tend to vary to a great extent with the lapse of time because the reaction proceeds even after the preselected period of reaction. Accordingly, this method requires the measurement of absorbancy or turbidity to be effected immediately after the predetermined period of reaction has lapsed; otherwise, variations in measured values will be so great that accuracy in measurement will be decreased. These difficulties will cause problems in the practical application of this method to the actual quantitative measurement of an antigen or antibody.
In conventional quantitative methods, the reaction of a sensitized carrier particle suspension and a sample solution often may be carried out with stirring in order to accelerate the reaction. However, aggregates formed by the antigen-antibody reaction may be decomposed easily by mechanical impacts or stimula when they exceed the binding power in the aggregates produced by the antigen-antibody reaction. Furthermore, the extent to which the aggregates are decomposed varies greatly with stirring conditions so that accuracy or sensitivity in measurement will be decreased.
In order to provide a spectrophotometric procedure having improved sensitivity and accuracy in determining an antigen or antibody to be determined in a sample solution, extensive studies have been made with the assumption that various problems and difficulties associated with conventional spectrophotometric techniques would be overcome if the agglutination reaction or the agglutination inhibition reaction were ceased at an appropriate stage of reaction and aggregates formed by the reaction were fixed, thereby preventing them from decomposing upon encountering mechanical means such as stirring.
As a result, it has been found that the employment of a fixing compound can cease the agglutination or agglutination inhibition reaction at a desired stage of reaction and prevent the aggregates formed thereby from being decomposed.